1. Field of the Invention
The present invention relates generally to the fields of biochemical pharmacology and drug discovery. More specifically, the present invention relates to discovery of compounds that inhibit bacterial NAD+-dependent DNA ligase.
2. Description of the Related Art
DNA is susceptible to damage caused by errors committed during replication and by environmental factors such as radiation, oxidants, and alkylating agents. The repair and replication pathways converge on a common final step in which the continuity of the repaired DNA strand or the replicated lagging strand is restored by DNA ligase, an enzyme that converts nicks into phosphodiester bonds. Nicks are potentially deleterious lesions that, if not corrected, may give rise to double-strand breaks which are themselves overtly catastrophic if not repaired by homologous recombination or ligase-mediated non-homologous end-joining. Accordingly, a complete loss of DNA ligase function is lethal in every organism tested.
DNA ligases catalyze the sealing of 5xe2x80x2 phosphate and 3xe2x80x2 hydroxyl termini at nicks in duplex DNA via three sequential nucleotidyl transfer reactions. In the first step, attack on the xcex1 phosphorus of ATP or NAD+ by ligase results in release of pyrophosphate or nicotinamide mononucleotide (NMN) and formation of a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide bond to lysine. In the second step, the AMP is transferred to the 5xe2x80x2 end of the 5xe2x80x2 phosphate-terminated DNA strand to form DNA-adenylate (AppN). In the third step, ligase catalyzes attack by the 3xe2x80x2 OH of the nick on DNA-adenylate to join the two polynucleotides and release AMP (FIG. 1).
DNA ligases are grouped into two families, ATP-dependent ligases and NAD+-dependent ligases, according to the cofactor required for ligase-adenylate formation (1). The structures of ATP and NAD+ are depicted in FIG. 2. The ATP-dependent DNA ligases are found in eubacteria, bacteriophages, archaea, eukarya, and eukaryotic viruses. ATP-dependent ligases are exemplified by the bacteriophage T7 and Chlorella virus enzymes, for which atomic structures have been solved by X-ray crystallography (2, 3). The viral ATP-dependent enzymes consist of a xcx9c200 amino acid N-terminal nucleotidyl transferase domain and a 100-amino acid C-terminal OB-fold domain (FIG. 3). Within the N-terminal domain is an adenylate binding pocket composed of five motifs (I, III, IIIa, IV, and V) that define the polynucleotide ligase/mRNA capping enzyme superfamily of covalent nucleotidyl transferases (4). Motif I (Kxc3x97DG) contains the lysine nucleophile to which AMP become covalently linked in the first step of the ligase reaction (3, 5). Motifs III, IIIa, IV, and V contain conserved side chains that contact AMP and are essential for the nucleotidyl transfer reactions (2, 3, 6). The C-terminal OB-fold consists of a five-stranded antiparallel xcex2 barrel and an xcex1 helix. The OB-fold domain includes nucleotidyl transferase motif VI, which contacts the xcex2 and xcex3 phosphates of the NTP substrate (7) and which is uniquely required for step 1 of the ligase reaction (8).
The NAD+-dependent DNA ligases have been described only in eubacteria. Genes encoding NAD+-dependent ligases have been identified and sequenced from at least 60 eubacterial species. Every bacterial species encodes at least one NAD+-dependent DNA ligase (referred to as LigA). The NAD+-dependent DNA ligase LigA is essential for growth of E. coli, Salmonella typhimurium, Bacillus subtilis and Staphylococcus aureus (9-13). It is reasonable to think that LigA will be essential for growth of all bacteria. NAD+-dependent LigA enzymes are of fairly uniform size (647 to 841 amino acids) and there is extensive amino acid sequence conservation throughout the entire lengths of the polypeptides. The atomic structures of NAD+-dependent LigA enzymes of two species of thermophilic eubacteria (Bacillus stearothermophilus and Thermus filiformis) have been determined by X-ray crystallography (14, 15). The structure of full-length Tfi LigA reveals that NAD+-dependent enzymes contain a catalytic core composed of nucleotidyl transferase and OB-fold domains (FIG. 3). Although there is scant amino acid sequence similarity between NAD+ and ATP ligases, the tertiary structures of the catalytic cores are quite well conserved and the adenylate binding pocket of NAD+ ligases is composed of the same five nucleotidyl transferase motifs described originally in the ATP-dependent enzymes. The nucleotidyl transferase motifs of the NAD+-dependent ligases are highlighted in FIG. 4. A notable distinction between ATP and NAD+ ligases is that the NAD+ enzymes lack a recognizable counterpart of nucleotidyl transferase motif VI within their OB-fold domain. The catalytic core of Tfi ligase is flanked by a 73-amino acid N-terminal domain (Ia) and three C-terminal domains: a tetracysteine domain that binds a single Zn atom, a helix-hairpin-helix domain (HhH), and a BRCT domain (named after the C-terminus of the breast cancer gene product BRCA1).
No NAD+-dependent DNA ligase activity has been identified from an eukaryotic cellular source. However, recent reports of the genomic DNA sequences of two insect poxvirusesxe2x80x94Melanoplus sanguinipes entomopoxvirus and Amsacta moorei entomopoxvirusxe2x80x94identified an open reading frame in each virus that encodes a polypeptide resembling the eubacterial NAD+-dependent DNA ligases (16, 17). Alignment of the 532-aa AmEPV ligase-like protein to the Tfi, Bst, and Eco LigA enzymes reveals conservation of domain Ia, the nucleotidyl transferase domain (including the five catalytic motifs) and the OB-fold (FIG. 4) as well as the HhH domain (not shown). However, the AmEPV protein lacks the Zn finger and the BRCT domains that are present in all bacterial NAD+-dependent LigA enzymes. Given that individual cysteine of the Zn finger have been shown to be essential for the nick joining activity of bacterial ligases (18, 19), and the hypothesis that the BRCT domain plays an important role in DNA binding (1), it is of considerable interest to evaluate whether the insect poxvirus gene product is a DNA ligase and whether it uses NAD+ as a cofactor.
NAD+-dependent DNA ligases are attractive targets for drug discovery. NAD+-dependent ligases are present in all bacteria and are essential for bacterial growth in all cases studied. Moreover, they are structurally conserved among bacteria, but display unique substrate specificity and domain architecture compared to ATP-dependent DNA ligases. Therefore, inhibitors of bacterial NAD+-dependent DNA ligases would be outstanding candidates for effective broad spectrum antibiotic therapy.
A plausible strategy for drug discovery is to identify the structural components of bacterial NAD+-dependent DNA ligase that interact with the NAD+ substrate and then to isolate small molecules that recognize these components and thereby block the binding of NAD+ to bacterial DNA ligase. The drug-binding site on the NAD+ ligase would ideally be unique to, and conserved among, NAD+ ligases, but absent from ATP-dependent ligases. The prior art is deficient in methods of executing this strategy because structural components of bacterial ligase that interact specifically with NAD+ are not known. The present invention fulfills this longstanding need in the art.
The present invention is directed to the identification of compounds that inhibit bacterial growth by inhibiting the functions of bacterial NAD+-dependent DNA ligase. Specific structural components within structural domain Ia of NAD+-dependent DNA ligase that are important for the reaction of DNA ligase with NAD+ and that comprise a putative binding site for the NMN moiety of the NAD+ substrate are identified.
DNA molecules and expression vectors encoding DNA ligase enzymes that are defective in their reaction with NAD+, but are active in the ligation of pre-adenylated DNA nicks are disclosed herein. The present invention also includes host cells containing these expression vectors as well as isolated recombinant DNA ligase enzymes that are defective in their reaction with NAD+, but active in the ligation of pre-adenylated DNA nicks. These defective DNA ligases are termed NAD+-defective ligases.
The present invention also discloses methods of screening for compounds that bind to the NAD+ substrate recognition site of NAD+-dependent DNA ligase or compounds that inhibit the enzymatic activities of NAD+-dependent DNA ligase.